1

PromotersDNA sequences that control the efficiency, site and direction of transcriptional initiation

1. Experimental Approach to identifying promoter elements

a. Mutational Analysis• Promoter Bashing• Saturation Mutagenesis

b. Sequence Comparison• Consensus Sequences

2. Role of Pol II Elementsa. Core Promoter Elements

• TATA Box, Inr, DPE, BRE, MTEb. Proximal Promoter Elementsc. Distal Promoter Elements

3. RNA Pol I and RNA Pol III promoters

RNA Pol II promoter Elements

2

Promoter Bashing/Mutational Analysisaromatase cytochrome P450Cyp19 Flanking Region

Cyp19 Flanking Region Luciferase

C Kalbe, R Fürbass, M Schwerin and J Vanselow 2000

H2,E2H1,E1

Mutational Analysis

3

Saturation Mutagenesis of βglobin promoterManiatis’s Lab

Consensus Sequences

4

Classes of Promoter Elements• Core Promoter Elements – DNA sequences

that interact directly with Pre-initiation complex (GTF/RNA pol) during initiation.

• TATA Box, Inr, BRE, DPE, MTE

• Proximal Promoter Elements – DNA sequences within 200bp of start that increase rate of initiation but interact with activators instead of Pre-initiation complex. [typically very position dependent]

• GC box, CAAT box, etc

• Distal Promoter Elements (Enhancers and Silencers) – Distance and orientation independent

TATA box• Consensus TATAAA

• found -30 to -25 relative start• Minimal Promoter?

• Found in less that 50% of Pol II genes• More common is luxury genes, less common in house keeping

genes• Functions

– Determines location of start • Move TATA box, move start (Chambon experiment)

– Role in efficiency of initiation• Remove TATA box of many genes reduce transcription. (Maniatis

Experiment)

MTE

5

S1 extension and mappingAntisense probe labeled with radioactive phosphate

RNA isolated from cells

Hybridization

S1 nuclease

Electrophoresis

Tata box and start siteChambon’s Tata Deletion

6

Chambon’s Results

Initiator sequence• Consensus Py Py A N (T/A) Py Py

• A is +1 Nucleotide• Originally identified as consensus at start site

• Minimal Promoter?• May be able to direct low levels of transcription in isolation of other

elements. However, typically works in context of other elements.• May be the most common of Pol II elements

• Functions– Directs start site to specific nucleotide– Role in efficiency of initiation

MTE

7

Downstream Promoter Elements• Consensus G (AT) C G

• found +30 to relative start• Common in Drosophila

• Minimal Promoter?• Often found on genes lacking a TATA box.• Almost always in conjunction with Inr (DPE-Inr Unit)

• Functions– Role in efficiency of initiation

• Remove DPE or change position relative to Inr reduces transcription

MTE

Critical Spacing between DPE and Inr

ALAN K. KUTACH AND JAMES T. KADONAGA, 2000

8

TFIIB Recognition Element (BRE)• Consensus (G/C) (G/C) (G/A) C G C C

• Just upstream of TATA box (and someforms just downstream)• Most BRE found on TATAless promoters

(role on tataless promoters not clear)

• Minimal Promoter?• Binding of TFIIB.

• Functions– Role in efficiency of initiation

– Acts like a silencer when just with TATA box– Acts like an positive element when with TATA and Activator

Deng and Roberts, 2005

wt Wildtype BREd

df Mutant BREd

Gal4-AH – Activator bindingto distal Enhancer

BRE Analysis

9

Motif Ten Element

DCE – Downstream conserved element

Lim et al, 2004

Other Proximal Promoter Elements in Core Promoters

MTE

Classes of Promoter Elements• Core Promoter Elements – DNA sequences

that interact directly with Pre-initiation complex (GTF/RNA pol) during initiation.

• TATA Box, Inr, BRE, DPE, MTE

• Proximal Promoter Elements – DNA sequences within 200bp of start that increase rate of initiation but interact with activators instead of Pre-initiation complex. [typically very position dependent]

• GC box, CAAT box, etc

• Distal Promoter Elements (Enhancers and Silencers) – Distance and orientation independent

10

Modular PromotersSV40 Early

Thymidine Kinase

Histone H2B

Module Consensus FactorCAAT box GGCCAATCT CTFGC box GGGCGG SP1Octomer ATTTGCAT Oct-1/2

TATA Inr

Distal Promoter Elements• DNA sequences that influence rate of transcriptional initiation and

can function in either orientation and at varying distances from start site.

Enhancers – sequences that increase rate of transcription• Simple Enhancers – bound by one transcription factor.• Complex Enhancers bound by multiple transcription factors.

Silencers – sequences that reduce rate of transcription

• Play a very important role in regulation of transcription. [Other elements function more in steady state transcription]

• Function in the context of core promoter and proximal promoter elements.

11

Enhancer within Ig intron

Enhancer Distance and Orientation Independence

12

RNA Pol I Promoter

Tjian et al, 1986

RNA Pol III Promoters

13

Promoter Bashing of 7SK

Proximal Seq ElementDistal Seq Element